FLIPi/TRAIL treatment inhibits mammosphere formation and prevents self-renewal of mammosphere forming units. A, Following c-FLIP siRNAi (FLIPi) or control siRNA (SCi) transfection, cell lines were plated in low-serum non-adherent culture conditions in the presence or absence of 20 ng/ml TRAIL at a density of 4,000 cells per well (20,000 cells/ml). Pictures are representative of mammosphere forming units (MFU) observed. B, Mammospheres from three replicate wells per condition were counted following seven days culture (Passage 1). Mammospheres were dissociated using trypsin, passaged at a density of 2,000 cells/well (10,000 cells/ml) in the absence of TRAIL and counted after seven days culture (Passage 2). Results are calculated as a percentage of mammosphere forming units from the total number of cells seeded and are representative of three independent experiments. C, siRNA-transfected SKBR3 and BT474 cells were treated +/- 20 ng/ml TRAIL for 18 hours, surviving adherent monolayers were then assessed for aldefluor activity by flow cytometry. Graph represents the percentage of cells scoring positive for aldefluor activity in three independent experiments (* P < 0.01 vs. SCi control).